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Destruction Complex
Home Cellular Regulation Home The following section will be focused on the main destruction complexes that were discussed at length in the class (B-Cat, SCF and MCL1, and Cbl). β-cat Destruction Complex Paper Discussed: : Intestinal Tumorigenesis initiated by dedifferentiation and acquistion of stem-cell-like properties. Cell(2013) The Paper shows: #Increased presence of β-cat causes tumorigenesis #*Since it is a poliferation protien only utilized in early cell development #This inflammatory response is mediated by the Transcription factor NF-kB decreases the lifespan of the cell by being 'hijacked' by the cancer. Homologous Recombination: Inducable promotor *The researchers had to create a transgenic embyronic stem cell (ES) to be introduced into the blastocyst of a mouse specimen. *The ES was engineered by the use of Homologous Recombination so as to have the 3rd exon of the Ctnnb gene excised by CRE Recombinase (by flanking exon 3 with Lox-P sites). **Excision of this exon removes the Phosopholyation site for GSK3 on β-Cat., This prevents GSK3 from marking β-Cat for degredation. This leads to a constitutively active β-Cat and tumorgenesis. *They also engineered the transgene so that CRE (which is not endogenous to the mouse system)is created with a complexed Estrogen Receptor (ER) that causes excision of the 3rd exon. **The destruction of CRE is stopped when introduced to a tamoxifen environment. *The tamoxifen is introduced orally to the mice and is an antagonist to the estrogen receptor to induce CRE recomberase. *They now created mice with constitutively active β-Cat present in adult cells causing tumorigenesis (which is also inducable). *In an environment with increased β-Cat activity, the researchers noticed that the presence of NF-kB was greatly increased. **NF-kB is a transcription factor that is responsible for a immune response in the cell *The cancer that was induced by the constitutively active β-Cat was illciting an immune response. This immune response is hijacked by the cancer cells to utlize the wound healing response. *When they crossed mice that had the NF-kB signal (TnF) removed (noted by the genotype: B-cat c.a./TnF -/-) that the survival of the mouse increased. *When NF-kB is active it created a phenotype that casued dedifferentiation of cells back to a state of rapid division. GSK3 Complex *GSK3 is a protien that phosphorylates the Serine and Threonine residues of its target substrates. *The substrates are then recgonized by E3 Ligases for the degradation via the Ubiquitin pathway. *In the paper, the GSK3 unit is complexed with APC and AXII NF-kB *Nuclear transcription factor *NF-kB plays a role in the immune response of the cell system *Normally, NF-kB is held in the cytoplasm by being complexed with the IKB subunit (note that in the figure NF-kB is both the p50 and RelA subunits). *The follwoing figure shows the possible way in which NF-kB is freed from IKB and allowed into the cytoplasm Immunohistochemistry *Refers to the process of detecting antigens in cells of tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues *The tissue sample is first fixed and then frozen. The sample is then thinly sliced and laid on a glass slide for staining and observation. B-cat Immune Response (Wound Healing) In the paper researchers utilized this mechanism to illustrate the effects of constitutively active β-Cat on epithelial tissue. *The first figure compares wildtype tissue with tissue experiencing active B-cat. *In tissue samples with active B-cat, it was noted that large dark areas (coresponding to areas of high concentration of B-cat) occured at the crypts (lower section) of the tissue. *This leads to the idea that B-cat stimulated growth from the bottom of the tissue to the top. *It is also noted that the overall size between the compared tissues is greatly different. The tissue with active B-cat is much more larger compared to the wildtype thus showing further evidence of increased growth. *BrdU staining measures the rate of poliferation in the cell. Looking at sections C and G (wildtype and active B-cat respectively) one can see that there is an increased rate of poliferation in the cells with active B-cat. *Section D and H of the first figure are also staining for the presence of Myc. Ubiquitin Ligase System *This complex of proteins is responsible for the ubiquitation of substrates marking them for degradation into the cell. *The complex of proteins are broken down into the E1, E2, and E3 subunits, each with a unique role in degradation. * The E1 complex is the initial carrier of the Ubiquitin molecule (denoted as Ub). *The Ub is then passed to the E2 subunit. *The E2 subunit and the attached Ub molecule then complex with the E3 molecule. *The E3 moecule (example: SCF- Skp1-Cullin-Fbox) contains a F-box unit that recognizes a specific amino acid seqeunce on its substrate and attaches to that substrate. *The E3 ligase then facilitates the ligation of the Ub molecule from E2 to be attached to the substrate molecule. *This happens many times, giving the substrate several Ub tags making it recgonizable for degradation. C.A B-cat and E3 degradation * Normally B-cat is phosphorlyated by GSK3 making it recgonizable to the f-box (B-TRCP) and for degradation. *In the paper the researchers made this Transcription Factor constitutively active by homologous recombination - removing exon 3 of its gene. *Thus the transcription factor is no longer recgonized by the E3 ligase and is no longer marked for degradation. SCF E3 ligase and MCL1 (Bcl-2 family) Mcl-1 protein blocks the pore fomation by Bax/Bcl-2 in the Mitochondrial Permeability Transition (PPT). Myc a well known oncogene that heavily up regulates proliferation but also it is able to stimulate Bax to cause apoptosis. This is considered a fail safe mechanism for cells if Myc is up regulated it will cause apoptosis. But if the Bax pathway is blocked either at the IAPs at the caspases or the with Mcl-1 blocking the PPT release of Cyto c and Smac from the mitochondria. Mcl-1 (Bcl-2 pro-survival) Conserved Sequence *The F box domain (also in cyclin F) is a scaffold to E3 ubiquitin ligase. When Mcl-1 is recognized by the F-box scafford protein (SCF complex) and ubiquitinated by E2 ubiquitin conjuating enzyme. E2 has been ubiquitinated by E1 ubiquitin activating enzyme. When Mcl-1 has been polyubiquitinated (polyubiquitin chain) it is destroyed by proteasome. SCF complex *Skp1 *Cullin *F-box Fbw7 is the F-box that binds Mcl-1 in SCF complex to push the cell towards the intrinsic apoptosis pathway by preventing Mcl-1's inhibition of of Bax. This was determind by the knockout and knockdown experiments in the Inuzuka and collagees (2011). E3 Ubiquitin ligase is unable to target Mcl-1. Create a homologous recomberase Fbw7 knockout with Cre recomberase, Lck P sites in T Cells. Final test was to attempt rescue of Fbw7 phenotype in Lymphnodes with induced knockout for Fbw7. When Fbw7 was injected back into these cells reduce Mcl-1 levels. The result allows for cell proliferation through Myc and surivival through Mcl-1. Because both Myc and Mcl-1 are spared and there is no release of cyto c or caspases 9/3. GSK3 requires a Ser residue to phospholate forward (Phosphorylation hops forward from Ser to Ser). This phosphorylation can be trapted to inhibit GSK3 at Ser 9 by Akt (resulting in an increase in B-cat). Mcl-1 is phosphorylated at consensus sequence and this allows the binding of Fbw7. If Ser residue is changed to Ala there is no assosication of Fbw7. A GSK3 inhibitor can block the assisociation of Mcl-1 and Fbw7. Apoptosis FACs anaylsis *ABT-737 is a bcl-2 antagoist (Apoptosis) *7 AAD DNA binding (dye) *Annexin (anti-inflammitory) References Adams J.M., Cory S. 2007. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 26: 1324 - 1337. Inuzuka H., Shaik S., Onoyama I., Gao D., Tseng A., Maser R.S., Zhai B., Wan L., Gutierrez A., Lau1 A.W., Xiao Y., Christie A.L., Aster J., Settleman J., Gygi S.P., Kung A.L., Look T., Nakayama K.I., DePinho R.A., Wei W. 2011. SCFFBW7 regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature. 471: 107 - 109. Welcker M., Clurman B.E. 2008. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nature Rev. Cancer 8: 83 - 93. Non-Fbox dependant E3 ligase: Cbl The following degradation pathway deals specifically with the removal of plasma membrane bound Tyrosine receptors. The degradation mechanism that is used does not contain an F-box recgonition, but rather recgonition of the receptor by an SH2 domain. Membrane Bound Receptors The membrane is home to thousands of receptors that are the beginnings of cellular signalling pathways. One important group of receptors is the family of receptors whose cytoplasmic tyrosines are phosphorlyated to begin signalling pathways. These families are important in the Cbl degradation pathway. Membrane bound receptors are in constant movement on the membrane, being shuttled in and out by vesicles to either be recylced or destroyed. It is the blockage of the receptors ability to be recycled back to the membrane which is key in Cbl degradation. Tyrosine Membrane receptors A class of membrane bound receptors who, when in contact with their specific extracellular ligand, become phosphorylated at their cytoplasmic residues. The phosphorylated residues can then phosphorylate other cytoplasmic proteins creating mutiple signalling pathways to occur. Control of these receptors occurs specifically at the phosphorylated tyrosine residues. SH2 domains on various proteins are responsible for the modulation of these receptors. SH2 domain and Tyrosine Receptors SH2 domiains are highly conserved protein domains found in many cellular protiens. The SH2 domain specifcally targets and docks with phosphorylated tyrosine residues. They are commonly found in protiens with key roles in tyrosine kinease pathways. Example: GRB2 is an SH2 protein which brings SOS to the membrane of a cell. *SOS is a GEF (Guanine Nucleotide exchange factor) that interacts with the GTPase RAS causing it to disassociate with GDP and associate with the highly abundant GTP in the cellular cytoplasm. *Association between Ras and GTP causes the activation of RAS and the interaction with RAF --> MEK --> MAPK --> TF AP1 With such an important role in signal transduction in the cell, how do we modulate or shut down this pathway? #Target the downstream protiens in the pathway (such as MAPK, AP1, MEK, etc.) #Turn off RAS by allowing it to associate with GDP rather than GTP #Remove the Ligand #Adding competitive SH2 domains to the specific Tyrosine receptor #Degrad the receptor in question Methods 1-4 offer some method of control but method 5 is by far the most simplistic and, thus, easiest to accomplish in the cellular environment. Method 5 is done through the help of Cbl. Cbl Ubiquitin Ligase Cbl is a E3 ligase that interacts and degrades its substrate without the use of an F-box and the corresponding recognition seqeunce. Degradation is done by the use of the RING domain located in the Cbl protein. The Cbl protien contains the following important protein regions: #Tyrosine Kinase Binding Domain #*Determines the traget substrate #RING Domain #*Location where the E2 subunit binds to Cbl #Proline Rich domain #*Location where SH3 proteins can bind to Cbl Cbl and Tyrosine Receptor interaction The SH2 domain (Tyrosine Kinase Binding Domain) of Cbl associates with the phosphorylated tyrosine residue of the membrane bound receptor. Associating with the receptor, Cbl is also bringing an E2 ligase that is associated to Cbl's RING domain. The proline domain of Cbl is also targeted by GRB2'S SH3 domain to form a complex.Since GRB2 also contains a SH2 domain, it too will associate with the membrane bound receptor at its second phosphorylated tyrosine residue. This creates a strong bivalent association with the receptor. When bound to the receptor, the Cbl/GRB1 complex is able to attach Ub to the receptor target. When poly-ubiquitinated the receptor is recgonizable to degradation.